A proposed general hypothesis of carcinogenesis consists of the following features: All cells possess multiple structural genes capable of coding for transforming factors which can release the cell from its normal constraints on growth. In adult cells, these structural genes are suppressed by diploid pairs of regulatory genes and some of the transforming genes are tissue specific. The structural genes loci are temporarily activated at some stage of embryogenesis and possibly during some stage of the cell cycle in adult cells. Spontaneous tumors, or tumors induced by chemicals or radiation, arise as the result of mutations of any set of regulatory genes releasing the suppression of the corresponding structural genes and leading to transformation of the cell. Autosomal dominant hereditary tumors, such as retinoblastoma, are the result of germ-line inheritance of one inactive regulatory gene. Subsequent somatic mutation of the other regulatory gene leads to tumor formation. Proc. Natl. Acad. Sci., 70:3324-3328 [1973]
Thus, under the above theory, it seems entirely plausible that cancer such as retinoblastoma may result when specific genes regulating certain types of cells are activated or inactivated. On the other hand, from the other available evidence outlined below, it is equally plausible that the retinoblastoma gene itself acts as one of the regulatory genes involved in tumorigenesis of certain tissues.
Retinoblastoma tumors have been associated with an interstitial deletions of the long arm of chromosome 13. The deletion of a specific locus on chromosome 13 would be followed by somatic mutation with the consequence of tumor formation. (Ibid).
Thus, the retinoblastoma tumor is a prototypic model for the study of genetic determination of cancer. Amer. J. Hum. Genet., 30:406-421 [1978].
Retinoblastoma, the most common intraocular cancer of childhood, occurs in both hereditarily and nonhereditarily acquired forms. The former is characterized by early age onset and multiple tumor foci compared to the nonhereditary type, which occurs later on with a single, unilateral tumor. Proc. Natl. Acad. Sci., 68:820-822 [1971]; Hum. Gen., 52:1-67 [1979]; and Science, 223:1028-1033 [1984].
Retinoblastoma is a malignant embryonal tumor of the sensory layer of the retina that arises in the retina of one eye (unilateral) or both eyes (bilateral), and it is frequently multicentric. Retinoblastomas are characterized by small round cells with deeply staining nuclei, and elongated cells forming rosettes. They usually cause death by local invasion, especially along the optic nerves. The molecular mechanism of the formation of this tumor is unknown.
Although most retinoblastoma ("RB") patients survive the disease, the consequences of undiagnosed and untreated retinoblastoma are severe. Therefore, an understanding of the genetic aspects and early diagnosis of this disease is very important. Without the isolation, identification and sequencing of the RB gene, such understanding and early diagnosis is very difficult.
Retinoblastoma occurs unilaterally in about 65% of cases and bilaterally in the remaining 35%. The majority of patients have a negative family history for this tumor. Despite this, it has been estimated that 40% of the patients, including all of those with bilateral disease, have a hereditary basis to their tumor. The genetic form of retinoblastoma demonstrates the characteristic features of hereditary cancers. Children having retinoblastoma at an earlier age average up to 9-10 months, usually have bilateral disease. Gen Cytogen, 58:534-540 [1986].
The pattern of inheritance is autosomal dominant, with a risk of 50% that the child of an affected parent will receive the gene for retinoblastoma, and a 90% chance that he or she will manifest the tumor. There is an increased risk for the development of second primary malignancies, particularly osteogenic sarcoma and fibroblastoma, both within and outside the field of radiation therapy. Molecular genetic evidence has been reported that the development of retinoblastoma and osteosarcoma involves the specific somatic loss of constitutional heterozygosity for the region of human chomosome 13 which includes the RB 1 locus. Proc. Natl. Acad. Sci, 82:6216-6220 [1985].
In addition, the same cancers that occur frequently as second primary tumors in patients with hereditary retinoblastoma have been observed in their relatives.
Two-mutation hypothesis explains the occurrence of retinoblastoma in both a hereditary and a sporadic form with differing frequencies of bilaterality. It proposes that all retinoblastomas occur as a result of two mutational events. In the hereditary form the first mutation is germinal, and thus is present in all cells of the individual. Only a single second mutation in the somatic target cell is required for development of the tumor. Therefore, these tumors might be expected to occur at an early age and to be bilateral. In the nonhereditary form, both mutations take place post-zygotically in the same somatic cell, and thus, these tumors would more likely be unilateral and occur at a relatively later age. Cytogenetic and molecular genetic studies of patients with retinoblastoma have provided data that strongly support this hypothesis.
Susceptibility to hereditary retinoblastoma is transmissible to offspring as an autosomal-dominant trait from parent to child with 90% penetrance. Under the above hypothesis, the inherited gene alone is not sufficient for carcinogenesis because not everyone who receives it develops the cancer, and not every susceptible cell carrying it becomes transformed. Thus, another event, a second gene change, or "second hit" such as deletion or rearrangement of the gene, is necessary.
The retinoblastoma gene deletions have been directly linked to the retinoblastoma. JNCI, 71:1107-1114 [1983].
A constitutional chromosome abnormality in a patient with retinoblastoma, with a deletion of one of the D group chromosomes, was previously reported. The D group chromosome involved in retinoblastoma cases is chromosome 13, and the band common to all the deletions is 13q14. These data suggested that the retinoblastoma gene is located within this chromosomal band.
Gene dosage studies have demonstrated that the locus for the enzyme esterase D is also located in band 13q14, and is closely linked to the retinoblastoma gene. Thus, there is available a biochemical marker for small deletions of this region of chromosome 13. Most of the patients with this specific chromosomal deletion have other phenotypic abnormalities in addition to retinoblastoma. Although there is no classical phenotype, developmental delay, microcephaly, microphthalmia, and skeletal and genitourinary malformations have been reported.
The patients with constitutional deletions of chromosome band 13q14 clearly demonstrate the first germinal mutation. Evidence for a second somatic cell mutation has recently been published in Nature, 305:779-784 [1983]. Restriction fragment length polymorphisms were used to compare constitutional and tumor genotypes of patients with retinoblastoma. The results suggest that tumorigenesis may result from the development of homozygosity for a recessive mutant allele at the retinoblastoma locus. In other words, the patients inherited one mutant allele, and homozygosity occurred when the normal allele was lost by mitotic nondisjunction or mitotic recombination. Thus, although pedigree analyses of families with hereditary retinoblastoma are compatible with an autosomal dominant pattern of inheritance, molecular genetic studies support the hypothesis that the retinoblastoma locus is recessive, and that both alleles must undergo mutation before the tumor is expressed. Gen. Cytogen., 58:534-540 [1986].
The genetic locus determining retinoblastoma susceptibility was assigned to band q14 of chromosome 13, along with the gene for the polymorphic marker enzyme esterase D ("ESD"), by examination of cytogenetic deletions. Science, 208:1042-1044 [1980]. The close linkage of these loci was confirmed by studies of retinoblastoma pedigrees. Science, 219:971-972 [1983].
The retinoblastoma susceptibility locus was further implicated in nonhereditary retinoblastoma by observations of frequent chromosome 13 abnormalities in tumor karyotypes and reduced esterase D activity in tumors. Cancer Genet. Cytogenet., 10:311-333 [1983].
The proposition has been made previously that inactivation of both alleles of the RB gene located in region 13q14 evidenced by the reduced esterase D activity resulted in retinoblastoma. Such proposition was based in part on a case of hereditary bilateral retinoblastoma LA-RB69 in which both RB alleles located in close proximity of the esterase D gene were inferred to be absent. Science, 219:973-975 [1983]. Recently, however, the assumption that the absence of esterase D activity implied loss of both esterase D and RB genes has been disproved by finding a low but detectable quantity of esterase D protein and enzymatic activity present in tumor cells LA-RB69. Hum. Genet., 76:33-36 [1987].
Nonetheless, Cavenee et al., (Nature, 305:779-784 [1983]) found that chromosome 13 markers that were heterozygous in somatic cells often became homozygous or hemizygous in retinoblastoma tumors. Homozygous 13q14 deletions at the molecular level in 2 out of 37 retinoblastoma tumors were also reported in Proc. Natl. Acad. Sci, 83:7391-7394 [1986]. These experiments provided evidence that the proposed RB gene may function in a "recessive" manner at the cellular level, in distinction to the "dominant" activities of classical oncogenes as measured, for example, by transfection assays. Science, 235:305-308 [1987] and Cancer Res., 46:1573-1580 [1986].
Thus, absence or inactivation of the RB gene, mapped to the chromosome 13q14:11 region, is believed to be the primary cause of retinoblastoma. Science, 213:1501-1503 (1981), Science, 223: 1028-1033 (1984), Proc. Natl. Acad. Sci., 68:820-823 (1971), Nature, 305:779-784 (1980). Since little of the RB gene structure or function is known, its cloning has proved to be difficult. Discover, March:85-96 (1987).
There is now enough evidence available showing the genetic involvement in the development of retinoblastoma and its secondary tumors. However, the precise location of the RB gene, its identification, isolation and nucleotide sequence and cloning, as well as the amino acid sequence of its protein product, is necessary for early diagnosis and treatment but difficult to obtain. It would be therefor advantageous to have this information available to enable the development of diagnostic procedure and treatment.
The latest reports suggest that the RB gene may have a regulatory function and that its presence and normal function prevents the development of the retinoblastoma. On the other hand, absence, malfunctioning or inactivation of the RB gene causes the development of, or genetical predisposition and susceptibility, to the retinoblastoma and is believed to be the primary cause for both hereditary and acquired retinoblastoma, osterosarcoma and fibroblastoma and other cancers.
Therefore, to find a way of determining the genetic predisposition in the fetus or the susceptibility in later age of acquiring retinoblastoma, by determination of the normal or abnormal function of the RB gene, is of utmost importance for early diagnosis and/or possible treatment of retinoblastoma, ostesarcoma, fibroblastoma and other cancers through a genetic manipulation.
The research on genetics of the retinoblastoma has a long history. Discover, March:85-96 [1987]. The major turn in retinoblastoma occured in early 1980 when general radioactive DNA probes were prepared against specific regions of chromosome 13. These probes were instrumental in findings that the retinoblastoma patients have abnormalities at 13q14 region of chromosome 13. Am. J. Hum. Genet., 36:10-24 [1984].
The next important finding of the esterase D gene location in the close proximity of the retinoblastoma gene was made in 1984. Then walking along the chromosome from the esterase D gene was initiated, including the cloning, probing, and screening. This approach eventually led to the identification, isolation and sequencing of the RB gene as disclosed in this invention. Science, 235:1394-1399 [1987].
Independently, there were other attempts to isolate RB gene by preparation and random use of DNA probes for the q14 region, using cells from the eye tissue of a fetal eye and from the retinoblastoma tumor. This random approach resulted in the finding of two tumor cells of 2 retinoblastoma patient missing part of the chromosome 13. Apparently at least a fragment of the gene was identified by one of the randomly constructed and used DNA probes. Later on, it was determined that the fragment was expressed in normal fetal eye tissue but was not expressed in retinoblastoma. Although scientists at that point called the results a detection of the gene, there was, and still is not sufficient evidence available that the whole gene was indeed identified at that time. The discovered fragment was later cloned. Discover, March:85-96 [198].
Since the discoveries of the RB gene fragment, published in Nature, Vol.323:643-646 [1986], only disclosed the randomly obtained but not identified and sequenced piece of DNA, albeit probably a portion of the RB gene, it would be greatly advantageous to identify the exact nucleotide sequence of the whole RB gene, including the initiation and termination codons to identify the amino acid sequence of the retinoblastoma gene protein product and to provide the antibody against such protein. The specific anti-RB protein antibody would be usable as the diagnostic tool in diagnosing retinoblastoma and other secondary cancers associated therewith and for regulation of carcinogenicity.
The localization of esterase D and RB genes in the same chromosomal region provides an advantageous approach for evaluation of RB gene functioning, for discovery of RB chromosomal patterns, for cloning of the RB gene, for isolating the RB gene and identifying the RB gene sequence by chromosomal walking, using the esterase D cDNA clone as the starting point. The isolation, identification and cloning of esterase D was part of the current discovery and is the subject of pending U.S. patent application Ser. No. 091547 filed on Aug. 31, 1987, hereby incorporated by reference. The protein product of the RB gene, namely phosphoprotein ppRB.sup.110, was isolated and its amino acid sequence determined as a part of this invention. It is independently disclosed and claimed in copending U.S. patent application Ser. No. 098612, filed on Sep. 17, 1987, and is hereby incorporated by reference. The subject matter was also published in Proc. Natl. Acad. Sci., 83:6337-6341 and 6790-6794 (1986). Science, 235:1394-1399 (1987).
The identification of the exact RB gene location, isolation, identification, sequencing and cloning of the RB gene; the identification of the RB protein, and preparation of the specific anti-RB antibody would allow diagnosis and treatment of the retinoblastomas and their secondary tumors and other cancers regulated completely or partially by the RB gene.
It is therefore one object of this invention to identify and isolate the RB gene.
It is another object of this invention to determine the exact nucleotide sequence of the cloned RB cDNA derived from RB gene.
It is yet another object of this invention to prepare RB cDNA and to clone the same.
It is a further object of this invention to prepare appropriate RB gene probes.
It is a further object of this invention to provide a method for diagnosing a retinoblastoma and its related secondary cancers and susceptibility thereto and to provide treatment of retinoblastoma by genetic manipulation.
It is still a further object of this invention to provide a method for diagnosing a tumorigenicity of tissue regulated by the RB gene.